Microelectromechanical systems (MEMS) have received a great deal of attention in recent years due to their promise for miniaturizing systems for a variety of applications. One particularly interesting facet of MEMS technologies is the possibility of coupling solid state devices with biological components (Bio-MEMS) such as biomolecules, cells, and tissues for creating novel bioanalytical systems.
Bio-MEMS technologies present a unique opportunity to study fundamental biological processes at a level unrealized with previous methods. The capability to miniaturize analytical systems enables researchers to perform multiple experiments in parallel and with a high degree of control over experimental variables. This capacity allows a high throughput approach for studying a wide variety of problems in biology.
Skeletal muscles are highly differentiated organs whose primary function is to generate longitudinal force for locomotion. Anatomically, myotubes or myofibers are composed of densely packed proteins (myofibrils), mostly myosin and actin, organized into functional structures called sarcomeres. During force generation the distance between the interconnected sarcomeres decreases as myosin pulls on the actin filaments. The process is mediated by Ca2+ release from the sarcoplasmic reticulum and is known as the sliding filament theory of muscular contraction (Huxley 1975; Gordon et al. 2000). Adult skeletal muscle is composed of two distinct types of fibers, extrafusal and intrafusal. The extrafusal fibers are part of the force generating motor circuit while the intrafusal fibers form the muscle component of a stretch sensor. Extrafusal and intrafusal fibers differ morphologically, functionally, and by their neuronal innervation.
The general structure of the sarcomere is consistent among extrafusal muscle fibers. However, adult skeletal muscle expresses multiple isoforms of myosin heavy chain (MHC) protein. Each isoform exhibits distinct ATPase activity that alters the physiological properties of the sarcomere and the myofiber overall. MHC classes can be divided into three isoforms type I, type IIa, and type IIb (Walro and Kucera 1999).
Type I muscle fibers contact slowly relative to the other isoforms due to slow ATPase activity and are slow to fatigue due to high levels of mitochondrial enzymes that generate large amounts of ATP. They contain a large number of mitochondria and myoglobin which give them a distinctive red color and are, therefore, known as red fibers. These fibers rely on aerobic respiration for ATP regeneration and are responsible for sustained, tonic contraction. They typically maintain an intracellular calcium level above 100 nM, but below 300 nM (Olson and Williams 2000; Scott et al. 2001). In vivo evidence suggests that chronic long term stimulation of fast twitch muscle fibers like the tibialis anterior causes a switch to the slow MHC isoform (Termin and Pette 1992; Pette et al. 2002). The integral membrane protein phospholamban is expressed exclusively in type I fibers where it regulates the Ca2+ pump adding an additional level of contractile rate control (Pette and Staron 2001).
Type IIa myofibers can be considered an intermediate between fast and slow twitch fibers. These muscle fiber types also contain a high number of large mitochondria as well as increased myoglobin levels, which also gives them a red appearance. However, they are able to split ATP rapidly which gives the myotubes a high contractile velocity. They are resistant to fatigue because of their high capacity to regenerate ATP by oxidation, but not as resistant as Type I fibers (Scott et al. 2001). The Ca2+ binding protein parvalbumin is expressed exclusively in Type II fibers where it aids in muscle relaxation by removing Ca230  from the cytoplasm of the myofiber (Pette and Staron 2001).
Type IIb fibers are known as white fibers due to their low levels of mitochondria and myoglobin. They also possess few blood capillaries and consequently rely on anaerobic respiration for ATP regeneration. Type IIb fibers contain a large amount of glycogen and split ATP very rapidly. These factors leave these muscle fibers prone to fatigue. Fast twitch glycolytic fibers (type IIb) are used for sudden bursts of contraction and are characterized by brief, high-amplitude Ca2+ transients and lower ambient Ca2+ levels (<50 nM) (Olson and Williams 2000; Scott et al. 2001). It has been previously determined that the increased thyroxine (T4) and triiodothyronine (T3) levels in hyperthyroid animal models results in a conversion to type II fibers while hypothyroidism results in conversions in the opposite direction (Caiozzo et al. 1992). In vivo data has also established that calcineurin will de-phosphorylate Nuclear Factor of Activated T-cells (NFATs) which will allow them to translocate from the cytosol to the nucleus. Once in the nucleus, they bind to promoters and enhancers that activate slow fiber type formation (Schiaffino and Serrano 2002). Cyclosporin is an inhibitor of the calcineurin signaling pathway (Schneider et al. 1999). Akt 1 induces type IIb fiber formation and can be activated by platelet derived growth factor (PDGF) (Izumiya et al. 2008).
Intrafusal fibers reside in specialized sensory structures called muscle spindles. Morphologically, muscle spindles consist of two to twelve intrafusal fibers that are distinct from the extrafusal fibers in both structure and function. These unique fibers can be categorized morphologically as nuclear bag1, nuclear bag2 and nuclear chain fibers based on the location of their nuclei (Matthews 1964; Kucera 1982b). In nuclear bag fibers, the nuclei are clustered in an enlarged central region, while in nuclear chain fibers, the nuclei are arranged in a single row localized in the equatorial region (Kucera 1982a; Kucera 1983). The spindle fibers are also unique in that morphological characteristics such as striation and myofibril density vary proportionately with distance from the center (Kucera and Dorovini-Zis 1979). In fact, they are heavily striated at their polar regions indicating the presence of contractile sarcomeric units, a feature that decreases moving equatorially until it is nearly absent. This feature plays an important role in the sensitivity to stretch seen in fibers and consequently the nerve terminals that innervate them. Due to their unique role in sensory perception, these fibers express a distinct protein called α cardiac-like MHC at their equatorial region.
One tissue of particular interest with respect to a variety of diseases is skeletal muscle. Diseases affect skeletal muscle in different ways. Some diseases, such as amyotrophic lateral sclerosis (ALS), affect the stimulating inputs from the neuromuscular junction. Other diseases affect the muscle directly such as muscular dystrophy and muscular atrophy, which cause deterioration of the muscles' ability to generate force. Thus, it is advantageous to have a system that allows the real-time interrogation of the physiological properties of muscle as well as the controlled addition of exogenous factors for comparative experimentation. However, it is first necessary to be able to apply the measurements to statistical analysis with regard to physiological factors such as peak stress generated, time to peak stress, the time needed for the muscle to relax to half of the peak stress, and the average rate of stress generation. All of these factors give information about the condition of the muscle and can be compared to published values.
The present study outlines a novel method for performing real-time quantitative measurements of the physiological properties of cultured skeletal muscle using a Bio-MEMS device. Stresses generated by myotubes were measured using a modified Stoney's equation, which quantifies stresses generated by a thin film on a microcantilever with known physical properties. By this method it has been shown that it is possible to quantitatively measure stress on microcantilevers that are in agreement with values previously published in the literature for cultured skeletal muscle. Furthermore, a method for selectively seeding and coculturing neuronal and muscle cells on these devices using microfluidic chambers was developed. By this method it was possible to create a model for studying neuromuscular junction development and function. This work validates the use of this system as a foundation for a high-throughput Bio-MEMS device.